Temperature-responsive substrates have been used for various purposes. For example, such substrates have been used as cell culture substrates, as explained below, as well as column chromatography fillers, systems of drug delivery (drug delivery systems, hydrogels, ion-exchange resins, membrane separation systems, desert-greening materials, water- and oil-repellents, and the like.
Culturing of adherent cells has been performed by attaching the adherent cells onto a glass surface or variously treated synthetic resin surfaces. For example, culture vessels having various surfaces, such as surfaces formed by hydrophilizing polystyrene by glow discharge or corona discharge, and surfaces formed by coating polystyrene with collagen or a biocompatible polymer, such as an MPC polymer, have been used (Patent Literature (PTL) 1, Non-patent Literature (NPL) 1).
To harvest cells cultured by attachment to the surface of such a material (a cell culture substrate), it is necessary to detach the cells from the surface of the cell culture substrate. To achieve this purpose, substances such as trypsin or like proteases or chemicals, having a function of disrupting binding between cells and the cell culture substrate, are used. However, enzyme treatments and chemical treatments are complicated, and are also noted to have disadvantages such as high probability of contamination and likelihood of impairing cells' natural functions due to their degeneration.
In recent years, in the field of regenerative medicine, 3D culture, etc., attention has been given to a sheet-like cell aggregate (a cell sheet), which is formed by attaching cells to each other through an extracellular matrix. However, a cell sheet, once formed on the surface of a cell culture substrate, has been difficult to harvest in the form of an intact cell sheet because enzyme treatments and chemical treatments disrupt the extracellular matrix, as well.
To overcome these drawbacks, there has been proposed a technique of detaching cells through temperature change by using a cell culture substrate coated with a temperature-responsive polymer having an upper critical solution temperature (sometimes abbreviated hereinafter as UCST) or a lower critical solution temperature (sometimes abbreviated hereinafter as LCST of 0 to 80° C. has been proposed (PTL 2). This temperature-responsive polymer-coated cell culture substrate not only allows harvesting of cells with less damage, but is also effective for producing a cell sheet.
However, there is leeway to further improve conventional temperature-responsive polymer-coated cell culture substrates in terms of cell detachability. More specifically, conventional temperature-responsive polymer-coated cell culture substrates have a problem in that a cell sheet may be broken when detached. This is attributed to the sea-island structure of temperature-responsive polymer coatings (PTL 3). Specifically, the island-shaped crystal portion is weak in cell detachment function because grafting does not easily progress in this portion, whereas the sea-like monomer solution portion is strong in cell detachment function because grafting readily progresses in this portion. When using such a temperature-responsive polymer-coated cell culture substrate, cells cannot be uniformly attached or grown. To solve this problem, optimizing irradiation conditions and drying conditions has been proposed (PTL 3).